Fig 2: Seh1 behaviour is highly correlated with the Nup107 complex and the small kinetochore-associated protein SKAP. (A,B) Scatter plot of H/L ratios (blue diamonds) of Nup107 (A) or TD60 (B) and Seh1 proteins across 38 different chromosome proteomic experiments plotted against each other. The value for Seh1 shows a strong correlation with that for Nup107 protein (Pearson correlation R=0.931) but no correlation with that for TD60 protein (Pearson correlation R=-0.208). (C) Correlation analysis for several chromosomal and kinetochore-associated proteins in this large set of experiments is presented as a color-coded matrix. The position of Seh1 on the matrix is shown by a black line. (D) Magnification of the Seh1 location of the matrix shown in C. (E) Profile plot showing the behaviour of SKAP (red line) following depletion of Seh1 across different experiments. (F) Control and SKAP-depleted cells were fixed and immunostained with anti-Seh1 (green) and -ACA (red) antibodies and for DNA (blue). SKAP depletion affects the kinetochore localisation of Seh1. (G) Quantification of Seh1 and Elys kinetochore levels in control (n=34 Seh1, n=30 Elys) and SKAP-depleted cells (n=35 Seh1, n=30 Elys) from three independent experiments. Fluorescence intensities are in arbitrary units (AU). ***P<0.0001 (two-tailed, unpaired t-test). Scale bar: 10 µm.

Fig 3: Seh1 depletion affects the association of the GATOR2 complex with mitotic chromosomes. (A) Seh1 depletion across different experiments only mildly affects kinetochore levels of Nup107 complex in chromosomes of DT40 cells. Profile plot showing the behaviour of Nup107 complex members (Nup133, Nup107, Nup85, Nup96, Nup160, Elys, Nup37, Nup43, Sec13) (red line). (B) Seh1 depletion strongly affects association of the GATOR2 complex with chromosomes in DT40 cells. Mio is shown as a red line while WDR24 and WDR59 are shown as blue lines.

Fig 4: Establishment of a rapid auxin-inducible Seh1 degradation system. Establishment of Seh1–mAIDmC HCT116 cells expressing OsTIR1 and Seh1–mAID–mClover. (A) Strategy for the insertion of the mAID–mClover coding sequence just upstream of the termination codon of the human Seh1 gene. A schematic diagram of the guide (g)RNA/targeting template-targeting site at Seh1 exon 8 is shown. The gRNA sequence is shown in green and PAM motif in red. The targeting template contains 500 bp homology arms that flank the double-strand break site and a mAID–mClover Hygro or/Neo cassette. The genomic configuration expected to be generated via homology-directed repair is shown underneath. (B) Schematic illustration of the AID system. OsTIR1 is part of the SCFOsTIR1 E3 ligase complex, and works together with an endogenous E2. Upon addition of indole-3-acetic acid (IAA), Seh1 protein tagged with mAID is polyubiquitylated and degraded. (C,D) The subcellular localisation of Seh1–mAID–mClover (green) is shown in interphase or mitotic cells in the absence or presence of IAA (4 h). Cells were counterstained for the Nup107 complex member (Elys, red), a nuclear envelope component (Lap2B) (red), and FG repeat nucleoporins (mAB414) (red). DNA is shown in blue. (E) Immunoblots of total cell lysates of from the HCT116 OsTIR1 parental cell line and Seh1-mAIDmC cell line in the absence (-IAA) or presence (+IAA) of auxin during interphase (E) or mitosis (F) probed using anti-Seh1 and anti-tubulin. Seh1–mAIDmC cells were synchronised in mitosis with Monastrol and were either mock treated or treated with IAA for the corresponding amount of time still in the presence of Monastrol. Tubulin serves as a loading control. *non-specific band (Seh1 is marked with a black dot). Scale bars: 10 µm.